The early development of vertebrates birds and mammals

The early development of vertebrates: birds and mammals 杨雪松 医学院 组胚系 & 生科院 再生医学教育部重点实验室 医学院 641， Tel： 85228316， E-mail: yang_xuesong@126. com

The e-mail for my lecture: ppt or any notices Development_2012@126. com Password: development

Human Embryo development movie

Developmental biology • Definition: Field of biology that studies how a single cell (the fertilized egg) gives rise to a fully formed organism. • Enjoyment: why do people get excited about the topic? Complexity, beauty, imaging, relevance (human congenital defects, cancer)

Developmental biology Description: Draw out embryonic development from 1 -cell stage, cleavage, blastula, gastrula (germ layer formation), body plan development, neurulation, organogenesis So most of what you have studied so far has been intracellular biology, focusing on molecules and what happens inside individual cells; one important aspect of biology that has not yet been covered is how cells communicate with each other, how they form multicellular organisms, how cells form different tissues, how these different tissues form organs that in turn interact with each other.

Fundamentals questions in developmental biology - Axis determination: AP, DV, LR. how do you ‘break’ the symmetry of the egg? Sperm entry, localized determinants Cell differentiation Cell proliferation Cell migration: mainly in gastrulation Cell polarization (symmetric vs asymmetric cell division), Cell shape (giving cells different morphologies, e. g. neurons, EMT), Cell death Morphogenesis (how cells come together to form tissues Organogenesis Germ cell development: fertilization Stem cells: multipotency, ability to self-renew, so ‘trendy’ Regeneration

Tools for developmental biology study • Gene knock-out or knock down: mutation, RNAi, antisense oligos - Gain-of-function experiment: m. RNA injections, transgenics - Biochemistry and molecular biology: Western blot, PCR… - Imaging: live, gene and protein expression - Experimental embryology: cell or tissue ablations, bead implantation, chick-quail chimeras

Model systems for developmental biology • Worm (C. elegans) • Fly (Drosophila melanogaster) • Fish (zebrafish, medaka) • Mouse • Xenopus (laevis, tropicalis) • Chick (Quail)

Model systems for developmental biology Chick: Gallus domesticus Fly: Drosophila melanogast er MODEL SYSTEMS for the experimental analysis of development Mouse: Mus musculis Fish: Danio rerio Xenopus laevis and tropicalis C elegans: Caenorhabdit is elegans

The early development of chick embryo

The developmental stage of chick embryo

The five fundamental main steps Gametogenesis fertilization cleavage gastrulation organogenesis

Modulation of cell migration in chick embryo gastrulation

Is cell migration the only event happen during gastrulation? Answer is No • Proliferation: divide 2 -3 time during gastrulation • Apoptosis However, comparing those… no doubt that cell migration is major event, at least in chick gastrulation

The advantage of chick embryo 1. Good model for human being, short life-span 2. Very easy to accessible and be manipulated. 3. Combination with Classic and molecular biological technologies. • • transplantation cell labelling Gene overexpression Gene knock-down Immunocytochemistry chemical treatment in situ hybridization

What is gastrulation? Gastrulation – the stage of animal development where a ball of cells consisting of a single, undeveloped layer is converted into an embryo consisting of three separate tissue layers : • Ectoderm – outer layer • Endoderm – inner layer surrounding the gut • Mesoderm – middle tissue layer

The first structure – primitive streak

Cell movement in chick embryo Gastrulation Hensen’s node primitive streak epiblast hypoblast migrating cells endoderm mesoderm displacing hypoblst

When does gastrulation happen ? List of developmental events in order: • • • Fertilization) Cleavage Blastocyst Grastrulation Neuralization

Primitive Streak Formation (elongation to anterior)

The main axes of a developing embryos

Specification of the chick anteriorposterior axis by gravity Rotation in the shell gland (A) results in the lighter components of the yolk pushing up one side of the blastoderm (B). That more elevated region becomes the posterior of the embryo (C).

Discoidal meroblastic cleavage in a chick egg (A-D) Four stages viewed from the animal pole (the future dorsal side of the embryo). (E) An early-cleavage embryo viewed from the side

Formation of the two-layered blastoderm of the chick embryo (A, B) Primary hypoblast cells delaminate individually to form islands of cells beneath the epiblast. (C) Secondary hypoblast cells from the posterior margin (Koller's sickle and the posterior marginal cells behind it) migrate beneath the epiblast and incorporate the polyinvagination islands. (D) This sagittal section of an embryo near the posterior margin shows an upper layer consisting of a central epiblast that trails into the cells of Koller's sickle (ks) and the posterior marginal zone (mz).

Streak formation in chick

Regulation of the chick blastoderm posterior marginal zone (PMZ) (Only Vg 1 expression PMZ Supplied by Xuesong Yang’s lab) The ability to initiate a primitive streak is found throughout the marginal zone. When the blastoderm is divided into four parts, each part can initiate gastrulation and give rise to an embryo.

The in vitro developmental methods for chick embryos New Culture EC culture Susan C etc. DEVELOPMENTAL DYNAMICS 220: 284– 289 (2001)

The microscopy for studying cell migration in gastrulation of chick embryos inverted Compound & Dissection Optical Systems Two-photon Confocal

Gene transfection …training required Just think of …. and keep trying…. .

Cell migration in gastrulation Cell movement pattern: 1. Epiblast cells movement during primitive streak formation. 2. Mesoderm formation during gastrulation. 3. Vasculogenesis Cell movement mechanism: 1. Cell-cell adhesive molecule. 2. Cell polarity. 3. 3. chemotaxis.

The correlation between morphogen and cells in pattern formation

Cell migration in gastrulation Cell movement pattern: 1. Epiblast cells movement during primitive streak formation. 2. Mesoderm formation during gastrulation. 3. Vasculogenesis Cell movement mechanism: 1. Cell-cell adhesive molecule. 2. Cell polarity. 3. 3. chemotaxis.

The genes involved in streak formation in chick

Convergent extension in Keller explants

The Wnt planar polarity pathway in convergent extension

Cell movement patterns in gastrulation

The role of Wnt 11 signalling

Streak formation as a result of intercalation and/or oriented cell division R Keller et al, 2003

Cell movements of the primitive streak of the chick embryo (A-C) Dorsal view of the formation and elongation of the primitive streak. The blastoderm is seen at (A) 3– 4 hours, (B) 7– 8 hours, and (C) 15– 16 hours after fertilization. The early movements of the migrating epiblast cells are shown by arrows. (D-F) Formation of notochord and mesodermal somites as the primitive streak regresses, shown at (F) 19– 22 hours, (E) 23– 24 hours, and (F) the four-somite stage. Fate maps of the chick epiblast are shown for two stages, the definitive primitive streak stage (C) and neurulation (F). The endoderm has already ingressed beneath the epiblast, and convergent extension is seen in the midline.

Migration of endodermal and mesodermal cells through the primitive streak (A) Scanning electron micrograph shows epiblast cells passing into the blastocoel and extending their apical ends to become bottle cells. (B) Stereogram of a gastrulating chick embryo, showing the relationship of the primitive streak, the migrating cells, and the two original layers of the blastoderm. The lower layer becomes a mosaic of hypoblast and endodermal cells; the hypoblast cells eventually sort out to form a layer beneath the endoderm and contribute to the yolk sac.

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Primitive Streak Regression (shorten to posterior)

Chick gastrulation roughly from 24 to 28 hours (A) The primitive streak at full extension (24 hours). The head process (anterior notochord) can be seen extending from Hensen's node. (B) Two-somite stage (25 hours). Pharyngeal endoderm is seen anteriorly, while the anterior notochord pushes up the head process beneath it. The primitive streak is regressing. (C) Foursomite stage (27 hours). (D) At 28 hours, the primitive streak has regressed to the caudal portion of the embryo. (E) Regression of the primitive streak, leaving the notochord in its wake. Various points of the streak (represented by letters) were followed after it achieved its maximum length. Time represents hours after achieving maximum length (the reference line, about 18 hours after incubation).

Formation of Hensen's node from Koller's sickle (A) Diagram of the posterior end of an early (pre-streak) embryo, showing the cells labeled with fluorescent dyes in the photographs. (B) Just before gastrulation, cells in the anterior end of Koller's sickle (the epiblast and middle layer) were labeled with green dye. Cells of the posterior portion of Koller's sickle were labeled with red dye. As the cells migrate, the anterior cells formed Hensen's node and its notochord derivatives.

Induction of a new embryo by transplantation of Hensen's node (A) A Hensen's node from a duck embryo is transplanted into the epiblast of a chick embryo. (B) A secondary embryo is induced (as is evident by the neural tube) from host tissues at the graft site.

Nobel prize was presented to the discover of Organizer

The change in cell shape in the neural plate during chick neurulation

The change in cell shape in the neural plate during chick neurulation

Hensen’s node regress & notochord extension 1. 2. 3. Possibility: addition extra cells by Hensen’s node, notochord and notochord plate cell division, mediolateral cells move into notochord 2. 3. Possibility: Convergence and extension of notochord maybe active process, which push Hensen’s node back posteriorly 3. Possibility: posterior blastoderm drive node regression and notochord extension 4. Possibility: Node actively pull itself posteriorly. Ray Keller et. Differentiation (2003) 71: 171– 205

Mesoderm cell fate map

What is mesoderm formation apart from gene expression? • How does the streak form? • Which signals control movement? • What is mechanism of movement? • Role of cell division • Chemoattractants/repellents (FGF/VEGF? ) • Adhesion molecules

EMT – the initial step for mesoderm cell formation Hensen’s node primitive streak epiblast hypoblast EMT migrating cells endoderm mesoderm displacing hypoblst (Epithelial-Mesenchymal Transition)

Cadherin-mediated cell adhesion during gastrulation Epiblast- epithelial cells – connected by E-Cadherin

EMT – the initial step for mesoderm cell formation Hensen’s node primitive streak epiblast E-Cadherin EMT N-Cadherin hypoblast migrating cells endoderm mesoderm displacing hypoblst

Cell migration in gastrulation Cell movement pattern: 1. Epiblast cells movement during primitive streak formation. 2. Mesoderm formation during gastrulation. 3. Vasculogenesis Cell movement mechanism: 1. Cell-cell adhesive molecule. 2. Cell polarity. 3. 3. chemotaxis.

Spatial-temporal regulation of PTEN and PI 3 K induces cell polarization in response to a chemo-attractant signal PI 4, 5 P 2 PI 3 K PTEN PI 3, 4, 5 P 3 Merlot & Firtel, (2003) JCS 116, 3471

Signalling to actin cytoskeleton PI 3 kinase/Pten Dictyostelium Dock 180 PH Gef Rac/cdc 42 Scar/wave Actin filaments Growth factor

Small GTPase – cell polarity

Cell migration in gastrulation Cell movement pattern: 1. Epiblast cells movement during primitive streak formation. 2. Mesoderm formation during gastrulation. 3. Vasculogenesis Cell movement mechanism: 1. Cell-cell adhesive molecule. 2. Cell polarity. 3. 3. chemotaxis.

Mesoderm cell migration pattern during gastrulation

The fate map of primitive streak cells

Somite formation in chick

The xxx eggs make me crazy everyday!!!! Could fully understand the student’s complain…

Many hypothesis for the mechanism of cell migration

Migratory cell contact inhibition

Migratory cell contact inhibition Supplementary Figure 1: Contact Inhibition of Locomotion controls Neural Crest (NC) directional migration in vivo a. When two NC cells collide they change their respective direction of migration. We show here that this process is dependent on the PCP pathway (symbolised here with yellow tees). b. In the embryo, NC cells migrate with high directionality (green arrow) in streams of limited width (represented as a white area). We showed that cell-cell contact is essential for this directionality. c. Cell polarization is inhibited by cell contact. This inhibition is controlled by the same mechanism that the one shown in panel a (yellow tees). d. Only leading cells are polarised as they have a free edge. Cells can only migrate in this direction and thusdirectionality is achieved.

Chemotaxis act as one of mechanism for directional cell migration during gastrulation

Questions • What is gastrulation? • What is the significance of gastrulation during embryonic development?